Project Summary Osteoporosis, low bone mass, and related problems such as fractures, are major public health problems with a substantial economic burden on our health care system. Bone is a dynamic tissue. In healthy bone, it is commonly believed that mesenchymal progenitors, including their most primitive form mesenchymal stem cells (MSCs), constantly generate bone surface osteoblasts and marrow adipocytes via sequential, unidirectional, and branched differentiation steps. Almost all types of osteoporosis are associated with diminished bone formation and increased marrow adiposity. Therefore, identifying true MSCs and illustrating their differentiation processes into osteoblasts and adipocytes are essential for understanding the disease mechanisms of osteoporosis. However, our current knowledge of MSCs and their descendant progenitors is mostly obtained by in vitro studies. The in vivo nature of these cells is still largely unknown. In the past, owing to a lack of proper in vivo investigative tools, we deliberately ignored the heterogeneity and plasticity features of mesenchymal lineage progenitors by simply referring to all progenitors as MSCs or mesenchymal progenitors and searched for one or a set of marker(s) to cover all of them. Recently, we discovered that in Col2-Cre Rosa- tdTomato (Col2/Td) mice, Td signal labels all endosteal (metaphyseal) mesenchymal lineage cells but no other cell types, thus providing a perfect system to comprehensively analyze the subpopulations of mesenchymal lineage cells from MSCs to mature cells. Most importantly, in the past two years, large scale single-cell RNA sequencing (scRNA-seq) technique has evolved so quickly that now it can solve cell heterogeneity in complex organs in a way that no other conventional approaches can achieve. After applying this technique to the top 1% Td+ endosteal bone marrow cells freshly isolated from 1-month-old Col2/Td mice, we identified 8 mesenchymal cell clusters and ordered them in a pseudotimeline trajectory that illustrates the divergent differentiation processes from true MSCs to osteoblasts/osteocytes, adipocytes, and chondrocytes. With this awesome power, we believe now is the right time to test our central hypothesis that progenitors in mesenchymal lineage cells are made of subpopulations in a continuum of states and that those subpopulations are maintained in a dynamic way for quick responses to intrinsic and extrinsic cues, such as aging and therapies. The following aims will be performed: 1) delineate and characterize the heterogeneous progenitor subpopulations of mouse bone marrow mesenchymal lineage cells; 2) investigate the effects of aging and PTH treatment on newly identified subpopulations of mesenchymal lineage cells. Our objectives are to delineate the entire in vivo differentiation process from true MSCs to mature cells and to unravel novel targets that promote new bone formation. Successful completion of this project will not only reveal critical features of MSCs and their descendant progenitors but also shed light on new directions to understand the disease mechanisms of osteoporosis and develop novel treatments.